Periodic Reporting for period 4 - TiMaScan (Recirculated tissue macrophages (TiMa) in blood: Novel approach to early diagnosis and treatment monitoring in oncology)
Okres sprawozdawczy: 2021-05-01 do 2022-10-31
Sensitive intra-tissue total body scanning is continuously performed by the monocyte/macrophage system. These cells are modulated by tumor-releasing factors and are actively recruited to the tumor sites where they engulf apoptotic/necrotic tumor cells. Once they have fulfilled their local tissue-cleaning task, they can migrate via lymph vessels to lymph nodes to present (tumor-) antigens to T-cells and potentially recirculate to the blood stream, where they can be monitored and evaluated for their phagolysosomal contents by flow cytometry.
Therefore, this project aims to unravel phagocytosis of cancer cells, their digestion into tissue-specific and/or cancer-related protein fragments, the migration/recirculation of tissue macrophages (TiMas) to blood, and the detection of intra-phagolysosomal protein fragments in blood TiMas by antibodies. Building on this information, flow cytometric scanning of blood TiMas (TiMaScan) will be developed into a novel tool for early diagnosis and treatment monitoring in oncology, focusing on colon, lung, breast and prostate cancer. TiMaScan diagnostics will be minimally-invasive (1-2ml of blood), rapid, accurate, broadly available and cost-effective, only requiring a flow cytometer. Also, it can be applicable for early diagnosis and disease monitoring in other medical conditions, in which tissue damage and/or inflammation plays a role (Figure 1).
Additionally, a new method was developed to evaluate the phagocytic ability (i.e. capacity of engulfment) of monocytes and dendritic cells. In line with this, and as we are interested in defining how phagocytic cells break down (digest) cancer cells into fragments, we are developing a new tool for isolation of subcellular compartments (lysosomes and phagolysosomes), where digestion takes place. This new method will allow for identification of the enzymes (proteases) involved in the digestion process to understand the digestion patterns of human monocytes, macrophages and dendritic cells and to reveal the cancer-derived protein fragments which could be potentially used for early diagnosis and treatment monitoring in oncology.
In short, due to extensive characterization of monocytic cells in multiple tissues (over 100 proteins evaluated), new subpopulations of monocytes were identified in peripheral blood of adult individuals. These populations are functionally different and behave differently in case of disturbed homeostasis (e.g. tumors or damage induced by surgery). For evaluation of the phagocytic ability of these new subsets, a new flow cytometry-based method was developed, that can also be used for other applications. The results of the extensive characterization of monocytes and other innate myeloid cells, also allowed the design of a novel single-tube flow cytometry panel for the identification of >20 innate myeloid cells in peripheral blood, and which can be employed for immune-monitoring of these populations in a single step, also in other clinical/diagnostic settings (Figure 3).
Furthermore, in order to characterize the whole proteome of the different monocyte, macrophage and dendritic cell populations, evaluation of the best strategy to analyse very low numbers of cells was performed, and a micro-method for identification of up to 900 proteins from 2,500 cells was established. In this context it should be noted that conventional proteomics uses millions of cells.
Overall, these results highlight the development of knowledge and technology required to successfully fulfil the objectives proposed for the next period of the TiMaScan project. Completion of these objectives will allow for the identification of the protease repertoire in monocytes, macrophages and dendritic cells, and therefore to improve the understanding of the processes involved in phagolysosome digestion and antigen presentation, knowledge that has applications beyond the scope of the TiMaScan project (e.g. vaccination). In addition, the new knowledge on normal tissue and tumor cells, resulting from the extensive characterization of these cell (transcriptomics and proteomics of purified cells) will provide a better understanding of the selection processes and disease (cancer) progression. Furthermore, if the TiMaScan concept proves to be correct and applicable in a clinical setting, a flow cytometry-based, minimally invasive method for cancer screening and monitoring will be developed, which has the potential for being cheaper and faster than the currently used imaging techniques and being applicable to other fields where tissue damage plays a role, such as neurodegenerative diseases and insidious infections.